1. Field of the Invention
This invention relates to a solution or a suspension of particles useful as a vaccine or vaccine intermediate. More especially, this invention relates to a process for the production of hepatitis B surface antigen (HBsAg) carrying particles in novel forms which are highly immunogenic. Still more especially, this invention relates to a process for inactivating an antigen containing mass where the inactivation is performed employing heat on a highly concentrated antigenic mass, to wit, one which has not been diluted by the addition of a protein thereto such as albumin. This invention relates to a process for the production of such an antigenic composition and to the use thereof for immunizing animals, especially humans.
2. Discussion of the Prior Art
The relationship between what is now referred to as the hepatitis B surface antigen (HBsAg) and hepatitis B virus was definitively identified many years ago by Alfred M. Prince (Pro. Nat. Acad. Sci (U.S.) 60:814-821, 1968). This antigen is primarily located on proteins embedded in the membrane of lipoprotein particles having a particle size of approximately 18 to 24 nm and filaments of a similar diameter. These are now known to represent fragments of membrane similar to that which surrounds the virion of HBV, also known as the "Dane" particle.
Thereafter, a vaccine containing such particles was disclosed in U.S. Pat. No. 3,636,191 by Blumberg et al. Prince and others thereafter disclosed other hepatitis B viral vaccines containing membrane proteins derived from Dane particles and filaments. These Dane particles and filaments contained or were associated with the hepatitis B e antigen found in many chronic carriers of the hepatitis B virus.
Since the work described above was conducted, a vaccine against the hepatitis B virus was introduced in the U.S. containing HBsAg particles. This vaccine is produced by enzyme digestion of HBsAg containing particles derived from the plasma of chronically infected HBV carriers, more or less as described in U.S. Pat. No. 3,636,191, supra. This vaccine is prepared by a costly purification process which results in substantial losses of its immunogenicity. As a result, a relatively large dose of the resulting antigen (HBsAg) must be administered to assure an adequate immune response. Because of these factors, the vaccine has been available only at a relatively high cost, about $30.00 per dose or higher. Since an original injection and two boosters are required, it presently costs approximately $100.00 to become immunized against the hepatitis B virus in the United States, a cost that is not affordable in those parts of the world where the need for this vaccine is greatest.
It has become desirable, therefore, to provide a hepatitis B vaccine containing hepatitis B surface antigen in a substantially pure form having greatly increased immunogenicity so that much smaller, less costly, doses can be used. It is, of course, also essential that the infective virus present in the starting plasma has been completely inactivated so as to present no risk of infection.
Hepatitis B infection affects, for the most part, individuals residing in developing countries in Asia and Africa where limited funds are available for public health measures. It is mandatory in this twentieth century to provide such a vaccine for the protection of the hundreds of millions of people who are at risk of infection by hepatitis B virus, and as a consequence suffer the risk of subsequent development of cirrhosis and liver cancer, and to provide this at a cost that is affordable. In many parts of the developing world this necessitates that immunization must cost less than $1.00 per person, if the vaccine is to be used.
In co-pending application Ser. No. 656,833 now U.S. Pat. No. 4,639,371, there is disclosed a process for the production of a vaccine or vaccine intermediate characterized by HBsAG containing particles which have been to a large extent transformed into forms having a novel morphology, never found in nature. These forms have surprisingly enhanced immunogenicity.
According to experiments therein reported, a concentrated HBsAg containing mass is obtained by the steps of:
A. Precipitating HBsAg from blood plasma, e.g., human blood plasma containing the same, by contacting the same with polyethylene glycol to separate HBsAg from other blood serum proteins contained therein;
B. Affecting negative adsorption of such separated HBsAg on hydroxylapatite to further remove the bulk of serum proteins;
C. Subjecting the so adsorbed HBsAg to isopynic centrifugation to separate remaining intact Dane particles, and to further remove the remaining traces of contaminating serum proteins;
D. Subjecting the particles so separated by centrifugation to heat inactivation by heating them while at a concentration of 0.5-10 mg/ml at a temperature of 101.degree. to 104.degree. C. for 2 to 5 minutes and thereafter cooling the heated particles, e.g., by introducing them into an ice water bath.
It was believed, however, that the heat inactivation step of step D, supra, needed to be done in the presence of stabilizing concentration of proteins to avoid denaturation of the particles.